All biosensors for determining the concentration of analytes in a sample of blood suffer from hematocrit sensitivity to some degree. The biosensor response decreases as the hematocrit of the sample increases. There is no single reason for this decrease in the signal, though some of the reasons include diminished diffusion of the analyte in the sample and increased solution resistance. One of the methods proposed for the elimination of hematocrit sensitivity is to filter the red cells from the sample. The membrane technology to filter red cells increases both the assay time and measurement imprecision. Oxygen sensitivity has presented a challenge. Biosensors employing the enzyme glucose dehydrogenase are not expected to be oxygen sensitive. However, the oxidation-reduction reactions of the mediator (or coenzyme) could involve free radical intermediates. When these intermediates have long lifetimes, molecular oxygen can quench them, thereby rendering the chemistry sensitive to oxygen tension.
U.S. Pat. Nos. 5,708,247 and 5,951,836 describe a disposable glucose test strip for use in a test meter of the type that receives a disposable test strip and a sample of blood from a patient and performs an electrochemical analysis. The working formulation comprises a filler, an enzyme effective to oxidize glucose, e.g., glucose oxidase, and a mediator effective to transfer electrons from the enzyme. The working formulation is printed over a conductive base layer to form a working electrode. The filler, for example, a silica filler, is selected to have a balance of hydrophobicity and hydrophilicity such that on drying it forms a two-dimensional network on the surface of the conductive base layer. The response of this test strip is claimed to be temperature independent over relevant temperature ranges and is substantially insensitive to the hematocrit of the patient.
In photometric biosensors, a membrane is typically used to separate red cells from a sample of whole blood. The use of a membrane increases the time of response. U.S. Pat. No. 6,271,045 describes a photometric biosensor that employs a correction method to compensate for hematocrit sensitivity. The biosensor comprises a support member that contains a spreading layer and a reagent layer, and a capillary tube in communication with the support layer and spreading layer for transporting a sample of body fluid thereto. A capillary tube is provided on the support member whereby a fluid containing an analyte to be tested is introduced into the tube and flows through the tube to the spreading layer and contacts the reagent layer. In order to compensate for hematocrit level in the case of whole blood, additional sensors can be implemented so that they inspect the capillary tube in the test device, one sensor at the beginning of the capillary channel and one at the end. In this biosensor, whole blood is applied to the capillary channel. The entry flow of whole blood is timed as it moves between sensors. The time that the blood takes to travel the length of the capillary tube is an indication of the hematocrit of the blood. That information is used to correct any shift in reflectance readings of the instrument caused by the hematocrit level. It is also known that the absorbance of hemoglobin can be measured, and the measurement can be used to account for the sensitivity of the measurement to hemoglobin.
The majority of electrochemical biosensors do not use membrane technology; hence, electrochemical biosensors suffer from hematocrit sensitivity. U.S. Pat. No. 6,284,125 describes a biosensor insensitive to hematocrit, where red cells are separated from plasma. U.S. Pat. No. 6,287,451 describes a biosensor that can employ a method in which hematocrit level can be measured electrochemically, and the corrected concentration of an analyte can be determined from the measured concentration of the analyte along with factors that depend on the sensitivity of the biosensor to hematocrit level. The magnitude of the hematocrit sensitivity is dependent on the type of biosensor and on the type of measurement. For example, if the reaction is allowed to go to completion, the lengthy reaction time allows for complete oxidation of the analyte in the sample, thereby making the measurement less sensitive to hematocrit.
U.S. Ser. No. 09/529,617, filed Jun. 7, 2000, incorporated herein by reference, describes NAD+-dependent and NAD(P)+-dependent enzymes having substrates of clinical value, such as glucose, D-3-hydroxybutyrate, lactate, ethanol, and cholesterol. Amperometric electrodes for detection of these substrates and other analytes can be designed by incorporating this class of enzymes and establishing electrical communication with the electrodes via the mediated oxidation of the reduced cofactors NADH and NADPH. NAD+-dependent glucose dehydrogenase can be used as the enzyme and 1,10-phenanthroline-5,6-dione isomer can be used as the mediator. This combination shows hematocrit sensitivity and oxygen sensitivity. The enzyme is not dependent on oxygen (oxygen does not act as a co-substrate as it does with glucose oxidase) and hence is expected to be insensitive to oxygen. However, the mediator reaction appears to be slow and hence is affected by the presence of oxygen. The mediation reaction involves free radical intermediates. If the reaction is slow, the free radical intermediates have longer half-life; hence, the probability of being quenched by molecular oxygen is high. Accordingly, the enzyme mediator combination shows oxygen dependency. The hematocrit bias of 1,10-phenanthroline-5,6-dione mediator is not clearly understood; however, it is speculated that the slow reaction rate of the mediator is responsible for significant hematocrit sensitivity. 4,7-Phenanthroline-5,6-dione does not exhibit as much sensitivity to variations in hematocrit or oxygen as does 1,10-phenanthroline-5,6-dione. However, the structure of 1,10-phenanthroline-5,6-dione renders it easier to synthesize than does the structure of 4,7-phenanthroline-5,6-dione. The starting materials for the synthesis of 1,10-phenanthroline-5,6-dione are much less expensive than are the starting materials for 4,7-phenanthroline-5,6-dione. Additionally, the reaction conditions for the synthesis of 1,10-phenanthroline-5,6-dione are much less severe than are the reaction conditions for 4,7-phenanthroline-5,6-dione. Accordingly, it would be desirable to reduce the sensitivity of 1,10-phenanthroline-5,6-dione to hematocrit sensitivity and oxygen sensitivity.
Glucose monitoring devices are calibrated at normal hematocrit. In samples having a lower hematocrit, the biosensor reads a higher than appropriate blood glucose level, and in samples having a higher hematocrit, the biosensor reads a lower than appropriate blood glucose level.